The present invention relates to proteins having transketolase activity, their use in test systems, and nucleic acids which code for these proteins.
Plants are able to synthesize organic compounds from atmospheric carbon dioxide using light energy with formation of oxygen. This process is called photosynthesis.
It is to be assumed that the efficient formation, utilization and distribution of the photosynthesis products severely affect the growth of a plant.
As plants are dependent on a functioning photosynthesis and comparable reactions do not occur in animal organisms, the photosynthesis apparatus presents itself as an ideal target for the use of herbicides.
The complex reactions which lead to carbon dioxide fixation are divided into light and dark reactions. The light reaction is used for making available energy in the form of ATP and reduction equivalents in the form of NADPH. In the dark reaction (reductive pentose phosphate cycle or Calvin cycle), these compounds are used for the synthesis of organic carbon compounds.
Some of the known herbicides (eg. dichlorophenylmethylurea or paraquat) act by inhibition of the light reaction. The dark reaction is not utilized as a point of attack for herbicides.
The enzyme reactions of the reductive pentose phosphate cycle are divided into three sections:
a) carboxylation PA1 b) reduction PA1 c) regeneration. PA1 (1) fructose-6-phosphate+glyceraldehyde-3-phosphate.fwdarw. erythrose-4-phosphate+xylulose-5-phosphate PA1 (2) sedoheptulose-7-phosphate+glyceraldehyde-3-phosphate.fwdarw. ribose-5-phosphate+xylulose-5-phosphate
In carboxylation, carbon dioxide reacts with the acceptor molecule ribulose bisphosphate (RuBP), whereby two molecules of 3-phosphoglycerate (3-PGA) are formed. After phosphorylation, 3-PGA is then reduced to glyceraldehyde-3-phosphate (GAP). In the regeneration phase, the acceptor molecule RuBP is resynthesized from the GAP formed. Of six molecules of GAP formed, one molecule can be employed for other metabolic pathways.
A multiplicity of the enzymes involved in the reductive pentose phosphate cycle are potential points of attack for herbicides. Plastid transketolase, however, assumes a special position. Like transaldolase, transketolase (E.C. 2.2.1.1.) catalyzes two reactions:
The substrates and products involved in the reactions represent points of linkage between the reductive pentose phosphate cycle and other metabolic pathways. Exported triose phosphates are used in the cytoplasm as substrates for glycolysis and gluconeogenesis. Fructose-6-phosphate is used as a precursor molecule for the preparation of starch in the plastids. Erythrose-4-phosphate is an intermediary between primary and secondary metabolism. Linked with phosphoenol pyruvate, erythrose-4-phosphate opens in the Shikimate pathway, which leads to the synthesis of aromatic amino acids and phenolic substances.
Ribose-5-phosphate is used as a substrate in different metabolic pathways.
In plant tissues, two transketolase isoforms were described which differ in their subcellular compartmentalization (Murphy and Walker, 1982, Planta 155, 316-320).
The plastid transketolase is responsible in green tissues for more than 75% of the total activity. The active enzyme is present as a homotetramer (holoenzyme) having a relative molecular weight of 150 kDa. As cofactors, transketolase needs vitamin B.sub.1 (thiamine pyrophosphate) and magnesium. In the absence of thiamine pyrophosphate or in the presence of mercaptoethanol, the tetramer dissociates into two dimers (apoenzymes) having a relative molecular weight of 74 kDa each. Holo- and apoenzyme are catalytically active, the holoenzyme having a substantially higher activity than the apoenzyme.